PROJECT SUMMARY/ABSTRACT Insulin resistance (IR) is a state of metabolic dysfunction that precedes Type II diabetes (T2D) and cardiovascular disease. IR is characterized by an impaired ability of insulin to promote plasma glucose disposal into insulin-sensitive tissue, the majority of which is skeletal muscle (SkM). Before insulin can stimulate muscle glucose uptake (MGU), it must first be physically transported from the plasma, across the continuous endothelium of SkM capillaries and into the interstitial fluid that bathes myocytes. In fact, the ability of insulin to stimulate MGU depends on the rate at which insulin crosses the capillary endothelium. Furthermore, preliminary data from our laboratory and others have demonstrated that insulin transport across the endothelium is impaired in the insulin resistant state. Despite the importance of insulin transport to insulin action, the mechanism of endothelial insulin transport and its long-term regulation in vivo are poorly understood. This proposal will focus on two Specific Aims to define the mechanisms that regulate insulin transport in vivo. These Specific Aims will test the hypotheses that (1) endothelial insulin transport is an active process that requires caveolar vesicles and the insulin receptor and (2) obesity-induced defects in insulin transport are rescued by exercise training through a nitric oxide(NO)-dependent mechanism. To study insulin transport in vivo, I have developed a highly innovative imaging technique to measure capillary permeability to a fluorescent insulin probe in live mice. This technique will be combined with conditional, endothelial-specific genetic modifications in mice to delineate the role of caveolin-1, the insulin receptor, and NO to insulin transport. The effects of these manipulations on SkM insulin sensitivity will be assessed using hyperinsulinemic-euglycemic clamps with isotopic glucose tracers in conscious, unstressed mice. The proposed studies will elucidate the mechanisms by which insulin transport is regulated acutely and in response to chronic stimuli (obesity and exercise). The results of these studies will improve the prospect of developing therapeutic strategies that target the vasculature and SkM insulin delivery in the treatment of T2D.